Tomasz Ołdak

Publications (7)10.45 Total impact

• Article: Direct use of the comet assay to study cell cycle distribution and its application to study cell cycle-dependent DNA damage formation.

  Marcin Kruszewski, Teresa Iwaneńko, Eugeniusz K Machaj, Tomasz Ołdak, Maria Wojewódzka, Lucyna Kapka-Skrzypczak, Zygmunt Pojda
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  ABSTRACT: The comet assay or single cell gel electrophoresis has proven to be a versatile and sensitive method of measuring the induction and repair of DNA damage in individual cells. However, one of the drawbacks of the assay is the bias caused by changes in the ability of cells to repair DNA damage in different cell cycle phases. Whereas the bias seems less important when G0 peripheral blood lymphocytes are studied, it might cause problems when proliferating cells are investigated. In this paper, we validate the assumption that the total comet fluorescence intensity corresponds to the position of the cell in the cell cycle and can be used to assign single cells to specific cell cycle phases. To validate the approach, we used a very homogenous blood mononuclear CD34(+) cell population in G0 phase (unstimulated) or stimulated to enter the cell cycle. An analysis of the cell cycle distribution revealed that the 15 comet intensity classes and the 100 comets usually analyzed in a typical comet experiment are sufficient to obtain a reliable cell cycle distribution comparable with the results obtained by the flow cytometry for the same cell population. The effect of the cell cycle position on the results obtained by the comet assay for proliferating and non-proliferating cell populations irradiated with 3 Gy of X-radiation is also discussed.
  Mutagenesis 04/2012; 27(5):551-8. · 3.18 Impact Factor
• Article: Inhibition of poly(ADP-ribose)polymerase does not affect the recombination events in CHO xrs6 and wild type cells.

  Maria Wojewódzka, Marcin Kruszewski, Tomasz Ołdak, Teresa Bartłomiejczyk, Aneta Goździk, Irena Szumiel
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  ABSTRACT: Activation of poly (ADP-ribose) polymerase -1 (PARP-1) is an early DNA damage response event that, together with phosphorylation of p53, prompts various cellular functions important in the maintenance of the genome stability. In mammalian cells, DSB are repaired by nonhomologous end-joining (NHEJ) and by homologous recombination (HR). To investigate the role of PARP-1 in HR, CHO-K1 wild type and xrs-6 mutant cell line were transfected with pLrec plasmids which carry two nonfunctional copies of the beta-galactosidase (lacZ) gene in a tandem array. In result of HR they can give rise to a functional copy of beta-galactosidase. To test whether PARP-1 affects the frequency of spontaneous and induced recombination repair, we treated CHO-K1 and xrs6 clones carrying chromosomally integrated pLrec with the PARP-1 inhibitor 3-aminobenzamide (3AB). Our results show that the spontaneous homologous intrachromosomal recombination frequency between the two lacZ copies was almost two orders of magnitude higher in xrs6 cells than in CHO-K1 cells, but that it was not affected by 3AB treatment. Induction of DNA damage by irradiation or electroporation of restriction enzymes did not significantly increase the recombination frequency. Furthermore, in both the cell lines, the effect of PARP-1 inhibition on DSB repair was examined using the neutral comet assay. There was no effect of 3AB treatment on DSB rejoining after 10 Gy irradiation. The results presented support the conclusion that PARP-1 is not directly involved in HR.
  Biophysik 12/2006; 45(4):277-87. · 1.70 Impact Factor
• Source

  Available from: Zygmunt Pojda

  Article: Nonviral transfection of human umbilical cord blood dendritic cells is feasible, but the yield of dendritic cells with transgene expression limits the application of this method in cancer immunotherapy.

  Sergiusz Markowicz, Joanna Niedzielska, Marcin Kruszewski, Tomasz Ołdak, Agnieszka Gajkowska, Eugeniusz K Machaj, Henryk Skurzak, Zygmunt Pojda
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  ABSTRACT: Dendritic cells (DC) generated from human umbilical cord blood might replace patients' DC in attempts to elicit tumor-specific immune response in cancer patients. We studied the efficiency of transfection of human cord blood DC with plasmid DNA carrying the enhanced version of green fluorescent protein (EGFP) as a reporter gene, to test if nonviral gene transfer would be a method to load DC with protein antigens for immunotherapy purposes. Cord blood mononuclear cells were cultured in serum-free medium in the presence of granulocyte-monocyte colony stimulating factor (GM-CSF), stem cell factor (SCF) and Flt-3 ligand (FL), to generate DC from their precursors, and thereafter transfected by electroporation. Maturation of DC was induced by stimulation with GM-CSF, SCF, FL and phorbol myristate acetate (PMA). Transfected DC strongly expressed EGFP, but transfection efficiency of DC, defined as HLA-DR(+) cells lacking lineage-specific markers, did not exceed 2.5%. Expression of the reporter gene was also demonstrated in the DC generated from transfected, purified CD34(+) cord blood cells, by stimulation with GM-CSF, SCF, FL, and tumor necrosis factor alpha (TNF-alpha). Transfection of CD34(+) cells was very efficient, but proliferation of the transfected cells was much reduced as compared to the untransfected cells. Therefore, the yield of transgene-expressing DC was relatively low. In conclusion, nonviral transfection of cord blood DC proved feasible, but considering the requirements for immunotherapy in cancer patients, transfection of differentiated DC or generation of DC from transfected hematopoietic stem cells provide only a limited number of DC expressing the transgene.
  Acta biochimica Polonica 02/2006; 53(1):203-12. · 1.49 Impact Factor
• Source

  Available from: Zygmunt Pojda

  Article: Nonhematopoietic stem cells of fetal origin--how much of today's enthusiasm will pass the time test?

  Zygmunt Pojda, Eugeniusz K Machaj, Tomasz Ołdak, Agnieszka Gajkowska, Marzena Jastrzewska
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  ABSTRACT: Stem cells originating at fetal age are for many reasons superior as a material for the regenerative medicine purposes, when compared to their adult counterparts. While hematopoietic cells, isolated from fetal liver or cord blood, have been well known for a long time and have passed practical tests as clinical transplantation material, the non-hematopoietic cells are newly recognized, and the knowledge of their phenotype and differentiation potential is rather insufficient. We, and the others, have identified a subpopulation of cord blood cells phenotypically different from hematopoietic cells (CD34-, CD45-, CD29+, CD44+, CD51+, CD105+, SH-2, SH-3), in vitro plastic adherent, and capable of multilineage differentiation. The other candidates for multipotential stem cells are cells extracted from umbilical cord or placental tissue. The preliminary observations suggest, that these cells, phenotypically similar to the nonhematopoietic cord blood cells, are capable of extensive replication in vitro and of multilineage differentiation into a variety of tissues including cardiac muscle, bone and cartilage, adipocytes, and nerve cells. The other possible medical applications include "rejuvenation" of selected tissues and systems in senile patients, and therapeutical cloning - for both purposes, cells at the fetal stage of genetic regulation may be more useful than cells collected from adult donors. There is still, however, a high level of uncertainty concerning future medical applications of fetal stem cells. Their numbers and characteristics may differ from the preliminary observations, and their behavior in vivo may not fulfill the expectations originating from the in vitro studies. Finally, the autologous applications of stem cells collected at the stage of birth may need the involvement of technical and financial resources for the storage of frozen cell samples throughout the period of life of their potential user. Such procedure seems possible from technical point of view, but may be inadequately substantiated by the eventual advantages.
  Folia Histochemica et Cytobiologica 02/2005; 43(4):209-12. · 0.81 Impact Factor
• Article: [Short term stimulation of megakaryopoiesis in cord blood derived hematopoietic stem cells in ex vivo model].

  Eugeniusz K Machaj, Agnieszka Gajkowska, Marzena Jastrzewska, Tomasz Ołdak, Marcin Kruszewski, Zygmunt Pojda
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  ABSTRACT: Ex vivo expansion of megakaryocytes may be useful to prevent the long-term severe thrombocytopenia following umbilical cord blood transplantation. This study aimed to establish the optimal conditions of short-term stimulation of megakaryopoiesis. Immunomagnetically isolated CD34 cells of umbilical cord were placed into serum free culture medium supplemented with combinations of growth factors as follow: Tpo (thrombopoietin) and IL-3 (interleukin 3); Tpo, IL-3 and SCF (stem cell factor) or Tpo, II-3, SCF and IL-11 (interleukin 11). The number of colonies of megakaryocytic cells (expressed CD41 antigen) were evaluated in clonal culture in 24-hours intervals up to 5 days. Besides the influence of used culture systems on the other hematopoietic lines was checked on the same time points. It was shown that the best results were obtained using combination of 4 cytokines. At 5-th day of expansion the number of CD41 positive cells increased in this warrant 8-fold. In case of erythroid or granulocyte-macrophage precursors the use of this combination of growth factors resulted in slightly elevated number of them. We can conclude that proposed model of expansion of megakaryopoiesis seems to be very effective and can to be useful for the shortening the post transplant thrombocytopenia period.
  Polskie archiwum medycyny wewnȩtrznej 11/2002; 108(4):959-64. · 1.37 Impact Factor
• Article: [Analysis of factors influencing the cord blood sample collection for clinical purposes].

  Ryszard Cesarz, Eugeniusz K Machaj, Tomasz Ołdak, Zygmunt Pojda
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  ABSTRACT: The survival rate of patients after cord blood transplantation depends on the number of nucleated cell transplants. The number of nucleated cells available for transplantation closely correlates with collected volume of cord blood. The influence of several obstetric factors on the volume of cord blood donation was investigated. Cord blood was obtained from 32 normal full-term deliveries. Length of gestation, age of mother, weight of placenta and length of umbilical cord were analysed for their impact on the volume of cord blood. Mean volume of collected cord blood was 103 ml. We did not establish the correlation between the volume of collection and the weight of placenta (measured after blood collection), the length of gestation and the age of mothers. A close and significant correlation (p < 0.05, r = 0.78) concerned the length of umbilical cord and the volume of cord blood donation. Length of umbilical cord is the parameter which may be useful for selection of the most promising cases in the collection of cord blood.
  Ginekologia polska 06/2002; 73(6):507-11. · 0.41 Impact Factor
• Source

  Available from: Zygmunt Pojda

  Article: Optimisation of transfection conditions of CD34+ hematopoietic cells derived from human umbilical cord blood.

  Tomasz Ołdak, Marcin Kruszewski, Eugeniusz Krzysztof Machaj, Agnieszka Gajkowska, Zygmunt Pojda
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  ABSTRACT: Human umbilical cord blood is frequently used as a source of transplantable hematopoietic cells and more recently as a target of gene therapy - a new approach for treatment of various disorders. The aim of our study was optimisation of the transfection conditions of cord blood-derived CD34(+) hematopoietic cells. Mononuclear cells fraction was isolated from cord blood samples by density gradient centrifugation. Subsequently, CD34(+) hematopoietic cells were separated on immunomagnetic MiniMACS columns. Pure population of CD34(+) cells was incubated in a serum free medium supplemented with thrombopoietin, stem cell factor and Flt-3 ligand for 48 h and then transfected with plasmid DNA carrying the enhanced version of green fluorescent protein (EGFP) as a reporter gene. We studied the influence of various pulse settings and DNA concentrations on the transfection efficiency, measured by flow cytometry as the fluorescence of target cells due to the expression of EGFP. The optimal settings were as follows: 4 mm cuvette, 1600 microF, 550 V/cm, and 10 microg of DNA per 500 microl. With these settings we obtained a high transfection frequency (41.2%) without a marked decrease of cell viability. An increase of the pulse capacitance and/or of DNA concentration resulted in a greater electroporation efficiency, but also in a decrease of cell viability. In conclusion, the results described here allow one to recommend electroporation as an efficient method of gene delivery into CD34(+) hematopoietic cells derived from human umbilical cord blood.
  Acta biochimica Polonica 02/2002; 49(3):625-32. · 1.49 Impact Factor